Telomere Maintenance in Laser Capture Microdissection-Purified Barrett's Adenocarcinoma Cells and Effect of Telomerase Inhibition In vivo

Department of Surgery, Wayne State University and Karmanos Cancer Institute, Detroit, Michigan 48201, USA.
Clinical Cancer Research (Impact Factor: 8.72). 09/2008; 14(15):4971-80. DOI: 10.1158/1078-0432.CCR-08-0473
Source: PubMed


The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo.
Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L.
Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model.
We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

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Available from: Jason Wong, Jan 17, 2014
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    • "Genomic DNA was isolated from captured cells using a silica-gel membrane based QIAamp DNA Micro Kit (Qiagen, USA) as described previously (Shammas et al., 2008) with modification. To prevent generation of abasic sites on the DNA and to minimize oxidative damage, 50 μM phenyl-tert-butyl nitron (SIGMA, USA) was supplemented (O'Callaghan et al., 2008). "
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    ABSTRACT: Telomere shortening is observed in peripheral mononuclear cells from patients with major depressive disorder (MDD). Whether this finding and its biological causes impact the health of the brain in MDD is unknown. Brain cells have differing vulnerabilities to biological mechanisms known to play a role in accelerating telomere shortening. Here, two glia cell populations (oligodendrocytes and astrocytes) known to have different vulnerabilities to a key mediator of telomere shortening, oxidative stress, were studied. The two cell populations were separately collected by laser capture micro-dissection of two white matter regions shown previously to demonstrate pathology in MDD patients. Cells were collected from brain donors with MDD at the time of death and age-matched psychiatrically normal control donors (N = 12 donor pairs). Relative telomere lengths in white matter oligodendrocytes, but not astrocytes, from both brain regions were significantly shorter for MDD donors as compared to matched control donors. Gene expression levels of telomerase reverse transcriptase were significantly lower in white matter oligodendrocytes from MDD as compared to control donors. Likewise, the gene expression of oxidative defence enzymes superoxide dismutases (SOD1 and SOD2), catalase (CAT) and glutathione peroxidase (GPX1) were significantly lower in oligodendrocytes from MDD as compared to control donors. No such gene expression changes were observed in astrocytes from MDD donors. These findings suggest that attenuated oxidative stress defence and deficient telomerase contribute to telomere shortening in oligodendrocytes in MDD, and suggest an aetiological link between telomere shortening and white matter abnormalities previously described in MDD.
    Full-text · Article · Jun 2014 · The International Journal of Neuropsychopharmacology
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    • "In follow-up studies, long term GRN163L exposure could limit the lifespan of cultivated cancer cells derived from glioblastoma [29], multiple myeloma [30] and Barrett’s esophageal adenocarcinoma [31] as well as breast [32], [33], lung [34] and liver [35] cancers. In mouse models, the inhibitor could inhibit the growth of xenografts produced in mice by the implantation of these human cancer cells [29], [30], [31], [33], [34], [35]. GRN163L is currently in clinical trials in patients with multiple myeloma ( "
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    ABSTRACT: Telomerase is required for the unlimited lifespan of cancer cells. The vast majority of pancreatic adenocarcinomas overexpress telomerase activity and blocking telomerase could limit their lifespan. GRN163L (Imetelstat) is a lipid-conjugated N3'→P5' thio-phosphoramidate oligonucleotide that blocks the template region of telomerase. The aim of this study was to define the effects of long-term GRN163L exposure on the maintenance of telomeres and lifespan of pancreatic cancer cells. Telomere size, telomerase activity, and telomerase inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer cell lines. The cell lines exhibited large differences in levels of telomerase activity (46-fold variation), but most lines had very short telomeres (2-3 kb in size). GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with IC50 ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1 (IC50 = 75 nM) and CD18 cells (IC50 = 204 nM) resulted in an initial rapid shortening of the telomeres followed by the maintenance of extremely short but stable telomeres. Continuous exposure to the drug eventually led to crisis and to a complete loss of viability after 47 (CAPAN1) and 69 (CD18) doublings. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX) and evidence of both senescence (SA-β-galactosidase activity) and apoptosis (sub-G1 DNA content, PARP cleavage). Removal of the drug after long-term GRN163L exposure led to a reactivation of telomerase and re-elongation of telomeres in the third week of cultivation without GRN163L. These findings show that the lifespan of pancreatic cancer cells can be limited by continuous telomerase inhibition. These results should facilitate the design of future clinical trials of GRN163L in patients with pancreatic cancer.
    Full-text · Article · Jan 2014 · PLoS ONE
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    • "More recently, Barclay et al. (2005) evaluated telomerase activity along with hTERT and splice variants in BE and adenocarcinoma and suggested a significant increase in telomerase activity occurring in patients with BE and adenocarcinoma. However, neither hTERT mRNA levels nor hTERT mRNA splicing patterns correlate with telomerase enzyme activity, as it depends on posttranscriptional and posttranslational modification of hTERT, which includes phosphorylation of hTERT protein, assembly into telomerase holoenzyme, and its association with other proteins such as hsp90 and p23 etc (Shammas et al., 2008). "
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    ABSTRACT: Background: Dysplasia and adenocarcinoma developing in Barrett's esophagus (BE) are not always endoscopically identifiable. Molecular markers are needed for early recognition of these focal lesions and to identify patients at increased risk of developing adenocarcinoma. The aim of the current study was to correlate increased telomerase activity (TA) with dysplasia and adenocarcinoma occurring in the setting of BE. Materials and methods: Esophageal mucosal biopsies were obtained from patients (N=62) who had pathologically verified BE at esophagogastroduodenoscopy (EGD). Mucosal biopsies were also obtained from the gastric fundus as controls. Based on histopathology, patients were divided into three groups: 1) BE without dysplasia (n=24); 2) BE with dysplasia (both high grade and low grade, n=13); and 3) BE with adenocarcinoma (n=25). TA was measured by a PCR-based assay (TRAPeze® ELISA Telomerase Detection Kit). Statistical analyses were performed using one-way ANOVA and post-hoc Bonferroni testing. Results: TA was significantly higher in biopsies of BE with dyplasia and BE with adenocarcinoma than in BE without dysplasia. Subgroup analyses did not reveal any significant correlations between TA and patient age, length of BE, or presence of gastritis. Conclusions: Telomerase activity in esophageal mucosal biopsies of BE may constitute a useful biomarker for the early detection of esophageal dysplasia and adenocarcinoma.
    Full-text · Article · Feb 2013 · Asian Pacific journal of cancer prevention: APJCP
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